The present invention relates to a method for efficiently producing L-glutamic acid by a continuous fermentation method. L-Glutamic acid is widely utilized as a food material such as seasoning and a raw material of various chemical products.
Following the increase of the demand for L-glutamic acid in recent years, the development of an industrially advantageous production method thereof has been desired, and the production methods thereof as described below have been invented.
(1) Batch culture method, Fed-batch culture method PA0 (2) Continuous culture method
Compared with the continuous culture method, these culture methods are advantageous in that the equipment therefor is simple and the cultivation is terminated for a short period and that damage due to contaminated bacteria is less. However, the concentration of L-glutamic acid in the culture broth is increased over time, and the productivity and yield thereof are decreased under the influence of osmotic pressure or inhibition due to the products. Thus, it is difficult to maintain stably high yield and high productivity for a long time.
The continuous culture method is characteristic in that a high yield and a high productivity can be retained for a long time, by avoiding the accumulation of an objective product at a high concentration in a fermentor.
The most simple and general mode of continuous culture method is a single-fermentor continuous culture method, and a great number of reports on the single-fermentor continuous culture method applied for amino acid fermentation, including L-lysine fermentation and L-arginine fermentation, have been issued (Lys: Toshihiko Hirao et. al., Appl. Microbiol. Biotechnol., 32, 269-273 (1989), Arg: Tomoki Azuma et. al., J. Ferment. Technol., 66 (3), 285-290 (1988)). During such fermentation of L-lysine and L-arginine, generally, the bacterial growth and the production of the objective amino acids simultaneously occur, and therefore, the single-fermentor continuous culture method has been readily applicable to these fermentation.
On the other hand, it has been considered that the application of the single-fermentor continuous culture method to L-glutamic acid fermentation is difficult. In other words, conventionally, it has been considered that L-glutamic acid cannot be accumulated at a higher level by such L-glutamic acid fermentation, unless the bacterial growth is terminated by any method including limitation of biotin which is an essential factor for the bacterial growth, addition of antibiotics such as penicillin, addition of surfactants and temperature change (1. Japanese Journal of Fermentation Engineering Association, Vol. 41, No. 12, 645-651 (1963)); 2. Recent Japanese Industrial Chemistry, Vol. 23, 121-142, Asakura Shoten). So as to produce a greater amount of L-glutamic acid by the batch culture method and the fed-batch culture method, the culture conditions should be set to terminate the bacterial growth. Thus, it has been considered that the fermentation of L-glutamic acid by the continuous culture method is unsuitable because the bacteria simply wash out under conventional culture conditions to terminate the bacterial growth, so that the bacteria cannot be maintained in the fermentor. As to research works of the continuous culture method for L-glutamic acid fermentation, hence, research works of multiple-fermentor continuous culture method comprising promoting the bacterial growth in a first fermentor and suppressing the bacterial growth in a second fermentor to produce L-glutamic acid, have been carried out at the early stage, such as those by Mr. Ueda, et. al. (Japanese Journal of Fermentation Engineering Association, Vol. 41, No. 9, 450-458, 1964) and Mr. Mimura, et. al. (Japanese Journal of Fermentation Engineering Association, Vol. 42, No. 2, 70-78, 1964).
Subsequently, two methods, namely a cell recycle culture method and an immobilized bacteria culture method, have been developed, as culture methods based on the concept that a process of bacterial growth and a process of producing L-glutamic acid should be independently carried out.
Firstly, the cell recycle culture method is a method comprising extracting the culture broth where L-glutamic acid is accumulated but the bacterial growth is terminated and separating L-glutamic acid from the bacteria to recycle only the bacteria (Unexamined Published Japanese Patent Application No. 52-136985; Unexamined Published Japanese Patent Application No. 60-133891 or Unexamined Published Japanese Patent Application No. 62-48394), and the method can maintain a high yield and a high productivity for a relatively long time. However, the method requires a system to separate the bacteria from the cultured broth, disadvantageously, so that the cultivation system is complex. Furthermore, because the bacteria terminating bacterial growth are recycled by the cell recycle culture method, the ability of the bacteria of themselves to produce L-glutamic acid is decreased over time, which limits the cultivation time.
Although the immobilized bacteria culture method can also recover L-glutamic acid at a high yield and a high productivity (H. S. KIM et. al., Biotechnology and Bioengineering, Vol. 26, 2167-2174 (1982)), on contrast, a carrier to immobilize the bacteria is expensive from the respect of practical application for the production of L-glutamic acid and additionally, the method has a great number of problems to be overcome, such as carrier exchange technique. Therefore, the method is not essentially suitable for industrial applications.
As has been described above, the culture conditions to produce L-glutamic acid at a high yield by the batch culture method and the fed-batch culture method require complete termination of the bacterial growth, and therefore, the ability of a bacterium to produce L-glutamic acid is decreased at the latter stage of culture, which causes difficulty in improving the productivity. By the conventional continuous culture method, additionally, a system is required to further add bacteria to a fermentor or to recycle the bacteria, and therefore, the method has many problems so as to attain industrial application as follows: the whole system is complex and the possibility of bacterial contamination is increased and that the high productivity can be retained with much difficulty because of cultivation under conditions to terminate the bacterial growth.
So as to meet the enlargement of the need toward L-glutamic acid and produce L-glutamic acid at a lower cost, the productivity of L-glutamic acid fermentation should be elevated at a higher level than conventional ones.